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Postexposure Prophylaxis with Zidovudine Suppresses Human Immunodeficiency Virus Type 1 Infection in SCID-hu Mice in a Time-Dependent Manner Chu-Chih Shih, Hideto Kaneshima, Linda Rabin, Reiko Namikawa, Polly Sager, John McGowan, and Joseph M. McCune

HIV Group, SyStemix, Palo Alto, California; Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

Occupationalexposure to the human immunodeficiency virus (HIV)has led to a lowbut finite incidence ofinfectionamonghealth careproviders. In such circumstances, postexposure administration of 3'-azido-3'-deoxythymidine (zidovudine; AZT) might be beneficial. Totest this possibility, the SCID-hu mouse(the immunodeficient C.B-17 scid/scid mouseengrafted with human hematolymphoid organs) was treated with AZT at different times after intravenous infection with a standard dose ofHIV (knownto infect 100% of animals). If givenwithin 2 h, AZT suppressed infectionin all animals; if givenafter 2 days, no suppression was observed. At least in someanimals, an AZT-sensitive phase lasted for as longas 36h. Thesedata support the hypothesis that prompt administration of AZT might be efficacious in suppressingacute HIV infection in humans. Further studies in the SCID-hu mouse might provide insight into treatment protocols of even greater efficacy.

Occupational exposure to humanimmunodeficiency virus (HIV) will continue to be a risk to healthcareproviders. Cumulative data suggest that transmission rates after a single exposure are lowerthan thoseof other bloodborne infections [1]. After infection, however, the chancethat AIDS will develop is high[2]. To prevent accidental exposures, theCenters for DiseaseControlhas urgedthe implementation of universal precautions [3]. Since accidental exposures will occur nonetheless, it has also been proposed that 3'-azido-3'deoxythymidine (zidovudine; AZT) be offered promptly to affected individuals [4]. Although no firmdata supportthe use of AZT after an exposure to HIV, postexposure therapy has been shown to modify retroviral infections in animal models. When AZT is givenwithin 4 h, infection of mice by Rauscher leukemia virus is suppressed [5]. Likewise, postexposure AZT prevented feline leukemia virus infection of cats [6]. It is notable that these studies are of uncertain relevance: not only are theyconducted withnonhuman retroviruses in the setting of nonhuman cells, but critical features of drug action and

Received 16 July 1990; revised 2 October 1990. Presented in part: HIV and AIDS: Pathogenesis, Therapy, and Vaccine, UCLA, Apri11990; VIthInternational Conference on AIDS,SanFrancisco, June 1990 (abstract FA 72). Financialsupport: NationalInstitutes of Health(AI-29323) and SyStemix. Reprints or correspondence: Dr. Chu-Chih Shih, SyStemix, Inc., 3400 W. Bayshore Rd., Palo Alto, CA 94303. The Journal of Infectious Diseases 1991;163:625-627 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6303-0032$01.00

metabolism withinthe host environment are likely to differ from those in humans. Extrapolation to humans is further challenged by a recent, well-documented report of seroconversion afteraccidental exposure to mv despite promptpostexposure AZT administration [7]. Isolated clinical data, likedatafromanimal models, should be viewed with caution. It is possible that AZT is not efficacacious afteran exposure; it is alsopossible thatit couldameliorate, if not prevent, the consequences of infection if given at optimal doses,bypreferred routes, or in combination with other antiviral compounds. However, given the low rate of infection and difficulties with patient accrual, clinical trials to investigate these possibilities are not likely. To circumvent theseproblems, wehave developed methods by which the humanhematolymphoid system (including fetal liver, bone, thymus, and lymph node) can be surgically implanted into the immunodeficient C.B-17 scid/scid mouse, creating the SCID-hu mouse[8]. The humanorgansare vascularized at the site of transplantation, and the cells within themdifferentiate andfunctionally interactas though in a human. When challenged with HIV, human CD4+ T and myeloid cells in the thymus and lymphnode are permissive for infection [9, 10]; CD4- human tissue (e.g., connective tissue) and endogenous murine organsare spared [11] (unpublished data). When the animals are given AZT before intrathymic infection with HIV, viral replication is later suppressed[12]. Thus,this is a smallanimalmodelin whichinfection of humanlymphoid organsby HIV can be studied in vivo. To address questions of antiviral efficacy, wedeveloped an assay in which SCID-hu mice can be challenged with stan-

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dardized stocks of free virus [11] (unpublished data). This assay is quantitative, time-efficient, and safe. It is highly reproducible: 2 weeks after inoculation of virus, the human lymph nodes in 100% of SCID-hu mice show signs of HIV replication. The assay also covers manyaspects of the interaction between HIV and the host in vivo: all stages of the viral life cycle can be observed, from entry into the bloodstream to movement into the parenchyma of a human lymphoid organ to subsequent infection of human CD4+ T and myeloid cells. Weused this in vivo model for acute HIV infection to address the problem of postexposure therapy with AZT. Materials and Methods Homozygous C.B-17 scid/scid mice weremaintained andconverted into SCID-hu mice as described [8]. In these experiments, human fetalthymus and mesenteric lymphnodes (18-23gestational weeks) wereused. Toexclude thepossibility ofprior infection by HIV, cells fromthe thymus weretestedfor the presenceof HIV by DNApolymerasechainreaction(PCR) (seebelow); if negative, lymph nodes fromthe samefetaldonor werecarefully dissected andtransplanted into the subcutaneous regionof the fourth mammary fat pad of the SCIDmouseundermethoxyflurane anesthesia. Virions fromthe infectious molecular cloneHN(JR-CSF) [13] wereprepared in a stock and quantitated with respect to tissue culture infectious doses on phytohemagglutinin-activated humanperipheral bloodmononuclear cells. For infection of SCID-hu mice, stock vials werequicklythawed andimmediately used.A volume of0.3 ml (corresponding to 120,000 TCIDso) was injected intravenously into the retroorbital plexus of anesthetized mice with a blunt-tipped 30-gauge needle. At various times after infection (0.5, 1, 2, 4, 8, 24, 36, and 48 h), AZT (250 mg/kg/day) wasadministered by intraperitoneal bolus injection followed by oral maintenance therapy (250 mg/kg/day) for a total of 2 weeks. Two weeks after inoculation of virus, the human lymph node implants of SCID-humice were analyzedby DNA PCR for signsof infection by HIV as described [12]. The total lymphocyte count in individual harvestedlymph nodes was lOs-H)6 cells. The PCR assay is sensitive enough to detectone HN-infected cell in lOS cells.

Results

In other experiments [11, 14], it has been determined that intravenous infection of SCID-humice that have lymphnode grafts occurs in a time- and dose-dependent manner. Injection of 120,000 lCIDso of HIV(JR-CSF) results in detectable infection of 100% of animalsat a 2-week end point. If AZT is provided in the drinking water for 1 day before infection, doses of 125-250 mg/kg/day completely suppress infection at 2 weeks. Usingthese data, protocols for postexposure therapywith AZT were designed. When SCID-hu mice were inoculated with 120,000 TCIDso of HIV(JR-CSF) and treated with AZT 48 h later (250 mg/kg intraperitoneally and then 250

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Figure 1. Suppression of humanimmunodeficiency virus (HIV) replication in SCID-humice infected with HIV and treated with an intraperitoneal injection(250mg/kg) of zidovudine (AZT) at indicatedtime after infection, then maintained on 250 mg/kg/day AZT in drinking water for 14 days.

mg/kglday orally),infection wasdetectable in all 2 weeks later (figure 1). This was the case even though AZT was first administered parenterally at high dose (fourtimes the dose required to protect 50% of animals). In contrast, if the same dose of AZT wasprovided at 0.5, 1, or 2 h after inoculation, signsof HIV infection were undetectable 2 weeks later. The extentof suppression diminished progressively as the dosing intervalexceeded 2 h: 80% of animals had undetectable levels of HIV if AZT was givenat 8 hand 40% at 24 h. Strikingly, 20% of the animals were free of detectableHIV even when AZT was given as late as 36 h after inoculation. Discussion

These results demonstrate that prompt postexposure therapy with AZT suppresses HIV infection of human lymph nodesin the SCID-hu mouse. If the antiviralcompound was givenwithin2 h ofinfection, viralreplication wassuppressed in all animals 2 weeks later. If given between 2 and 36 h, lessprotection was provided until,at 48 h, therewas no effect. Translation of these data into clinical practice should be madewiththe following caveats in mind. First, the assaysystem is stringent. Large inocula of virus are given to ensure 100% infection at 2 weeks. Such conditions will not occur often, if ever, in the workplace. Second, the end-pointmeasure of infection (PCR) has a finite sensitivity and may not detect a small number of infected cells. We havepreviously shown that whenthehumanthymus implantofSCID-hu mice is directly injectedwith HIV, some cells show signs of HIV infection at 2 weeks, even whenAZT coverage is started24 h before inoculation of virus [12]. These cells cannot be detected by PCR but can be detected by in situ hybridization; if AZT treatmentis stopped, viral spread can be observed. Thus, suppression of viral infection cannot be equated with

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complete protection against HIV infection of the SCID-hu mouse. Finally, theexactdegreeof concordance between data in theSCID-hu mouse and in humans is notknown. Ourresults do not indicatethat prompt administration of AZT will prevent HIV infection in humans, but they supportthe hypothesis that AZT, given quickly, will provide a beneficial effect. In addition, this studydemonstrates the power of the SCIDhu system in determining the efficacy of antiviralcompounds in vivo. Many variables can be manipulated, including the size of the viral inoculum, the dose of AZT, the route of administration (of virus or drug), the timing of drug administration, and the assay for drug effect, to attempt to enhance the effectiveness of the treatment under study. Thus, it may be found that postexposure AZT shouldfirstbe givenparenterally (insteadof orally,as in the recentcase report [7]), that higher doses wouldbe more efficacious, or that coadministration of another antiviral or immunomodulatory agentwould show synergistic effect. It is unlikely that such treatment criteria couldbe systematically manipulated in a humanclinical trial. Careful experimental analysis with the SCID-hu mouse may thus provide insight into optimal treatment programs later. Perhaps the moststriking finding ofthis study was thatAZT continued to suppress infection in some animals as long as 36 h after inoculation of virus. This indicates that an AZTsensitive phase of viral replication can persistfor at leastthat long after acute infection in vivo. It is possiblethat thisphase is relatedto theformation of a replicative intermediate ofHIV. Zack et al. [15] recently showed that HIV can enter and synthesize viral DNA in both resting and stimulated T cells. In restingbut not stimulated cells, viralgenomic RNAis incompletely converted into full-length double-stranded DNA. These replicate intermediates have an intracellular half-life of rvt day. Sincetheir completionrequires reverse transcription, the process may be inhibited by AZT. Thus, if resting target cells greatly outnumber stimulated cells (as may be the case in the human lymphoid organs of the SCID-humouse), HIV infection in most cells mightlead to the formation of labile, AZT-sensitive replicative intermediates. The extentanddurationofAZT protection mightthereby be enhanced. Second, if mitogenic stimuli move cells from a resting to an activated phase (as might occur in the setting of extraneousantigenic stimulation), the durationof the protectiveeffect of AZT prophylaxis shouldbe shortened.These predictionscan be directly tested in the HIV-infected SCIDhu mouse. Such studies may contribute not only to a more complete understanding of HIV replication in human lymphoid organs in vivo but also to better protocols for postexposure prophylaxis against HIV in humans.

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Acknowledgment We thank Tom O'Toole (SyStemix) for his careful help. References 1. Henderson DK. HN-l in thehealthcaresetting. In: Mandell GF,Douglas RGJr, BennettJE, eds. Principles and practiceof infectious diseases. 3rd ed. New York: John Wiley & Sons, 1989. 2. Hessol NA, Rutherford OW, Lifson AR, et al. The natural history of HN infectionin a cohortof homosexual and bisexualmen: a decade of follow-up [abstract4096]. In: Programand abstracts: IV InternationalConference on AIDS(Stockholm). Washington, DC: Bio-Data, 1988. 3. Centersfor DiseaseControl. Update: universalprecautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus,andotherbloodbome pathogens in health-care settings. MMWR 1988;37:377-88. 4. HendersonDK, GerberdingJL. Prophylactic zidovudine after occupationalexposure to the human immunodeficiency virus:an interim analysis. J Infect Dis 1989;160:321-7. 5. Ruprecht RM, O'BrienLG, Rossoni LD, Nusinoff-Lehrman S. Suppression of mouse viraemia and retroviral disease by 3'-azido-3'deoxythymidine. Nature 1986;323:467-9. 6. Tavares L, Roneker C, Johnston K, Nusinoff-Lehrman S, de Noronha F. 3'-Azido-3'-deoxythymidine in feline leukemiavirus-infectedcats: a model for therapy and prophylaxis of AIDS. Cancer Res 1987;47:3190-4. 7. LangeJMA, BoucherCAB, Hollak CEM, et al. Failureof zidovudine prophylaxis after accidentalexposure to HIV-1. N EnglJ Med 1990; 322:1375-7. 8. McCuneJM, Namikawa R, Kaneshima H, Shultz LD, LiebermanM, Weissman IL. The SCID-humouse: murine model for the analysis of human hematolymphoid differentiation and function. Science 1988;241:1632-9. 9. Namikawa R, Kaneshima H, Lieberman M, Weissman IL, McCuneJM. Infection of the SCID-hu mouse by HN-1. Science1988;242:1684-6. to. Namikawa R, Fedor J, Kaneshima H, McCune JM. Characterization of HIV-l infected cellswithinthehumanlymphoid organs oftheSCIDhu mouse[abstractThA296]. In: Programand abstracts: VIth International Conference on AIDS(SanFrancisco). Washington, DC: BioData, 1990. 11. Kaneshima H, Namikawa R, ShihCC, RabinL, McCuneJM. HIV infectionof humanlymph nodes in the SCID-humouse. In: Program andabstracts: VIthInternational Conference on AIDS(SanFrancisco) Washington, DC: Bio-Data, 1990. 12. McCuneJM, Namikawa R, ShihCC, RabinL, Kaneshima H. 3'-Azido3'-deoxythymidine suppresses HIV infection in the SCID-humouse. Science 1990;247: 564-6. 13. Koyanagi Y,MilesS, Mitsuyasu RT, Merrill JE, VintersHV, Chen ISY. Dual infection of the central nervoussystemby AIDS viruses with distinct cellular tropisms. Science 1987;236:819-21. 14. McCune JM, ShihCC, Namikawa R, Rabin L, Kaneshima H. The evaluation of antiviral compounds in the SCID-hu mouse [abstract FA 72]. In: Program andabstracts: VIthInternational Conference on AIDS (San Francisco). Washington, DC: Bio-Data, 1990. 15. ZackJA, Arrigo SJ, Weitsman SR, Go AS,HaislipA, ChenISY. HIV-l entryinto quiescent primarylymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 1990;61:213-22.